Generally, the production of DNA chips comprises the steps of producing a probe, and integrating the produced probe on a slide. Methods for producing the probe are classified into two categories according to the base sequence length of the probe. Namely, if the probe is an oligonucleotide consisted of 15-25 bases, it is preferably produced by a chemical synthesis method using a synthetic group, etc., but if it is a DNA consisted of more than 100 bases, it is preferably synthesized by PCR amplification. However, the former has a shortcoming that a probe should be produced each time a DNA chip is produced, and the latter has a shortcoming that primers should vary depending on the sequence of a probe.
The present invention aims to provide a producing method of DNA chips, capable of overcoming such prior problems, and to produce DNA chips for the diagnosis of HPV (human papilloma virus) causing cervical cancer.
Cervical cancer refers to cancer that occurs at the uterine cervix. Cervical cancer is the most frequent gynecologic cancer in Korean women (about 6,000 new patients are diagnosed as cervical cancer annually), and is a disease requiring special care. It most frequently occurs in women in their late 40's, but recently, its attack in young age groups shows a tendency of an increase. If it has developed, it can be difficult to cure completely, but if it is diagnosed early, it can be completely cured by the modern medicine. Also, it is known that cervical precancer lesions occur before progression to cervical cancer, and thus, it is important to diagnose cervical cancer at the early stage.
Although the cause of the development of cervical cancer is not yet completely explored, but HPV is being noticed as the most important cause factor. Some of HPV infections are progressed to cervical cancer via precursor lesions such as high grade squamous intraepithelial lesion (HSIL), Carcinoma in situ (CIS), and this process is known to occur over a significant period of time. Thus, the detection of HPV infection with PAP smear, colposcopy, or cervicography, etc., which is known for early diagnosis method of uterine cancer will greatly help early diagnosis of cervical cancer.
Methods for detecting HPV infection and genotype can be broadly divided into an in situ detection method for HPV DNA, and a method using HPV DNA amplification. Examples of the in situ detection method for HPV DNA include liquid hybridization (hybrid capture by digene diagnostics, Silver Spring, Md.), Southern blot and dot blot using HPV type-specific probes, and filter in situ hybridization (FISH).
Examples of the method using HPV DNA amplification include type-specific PCR, and general primer PCR. The genotypes of HPV DNA amplified with a general primer set can be detected by various methods, including dot blot hybridization, microtiter plate hybridization and line probe assay. The line probe assay is a method for detecting about 20 genotypes using oligonucleotide probes immobilized on a nitrocellulose membrane, but has several problems in view of probe sensitivity and data analysis.
Furthermore, a commercial hybrid capture kit can detect HPV DNA without PCR by easily isolating HPV DNA from a clinical sample. However, this kit makes it possible to only determine whether the corresponding HPV DNA belongs to high-risk group or to low-risk group, and it is impossible for this kit to determine an accurate genotype. Thus, there is a problem in that noteworthy HPV genotypes (HPV 16 and 18) among the high-risk groups, i.e., genotypes having a very high correlation with the generation of cancer, cannot be distinguished from other high-risk groups (medium risk groups). Moreover, due to the use of RNA probes, this method has other problems in that stability is low and contamination possibility cannot be excluded.
In addition, there is another method for detecting HPV DNA, in which a chip on which about 50 bp single-stranded oligonucleotide derived from various HPV genotypes is integrated is brought into contact with a fluorescence-labeled DNA sample containing a base sequence specific to HPV genotypes, and then, HPV infections are determined depending on whether HPV DNA hybridization occurs or not. However, this method has a shortcoming that a process after preparing a chip is complicated as compared to a method using a DNA chip on which double-stranded DNA is integrated. Another shortcoming is that it is not easy for the DNA sample to access to the probe on the chip depending on the coating uniformity of a glass slide surface, so that the signal intensity after hybridization has no consistency.
In an attempt to solve the above-mentioned problems occurring in the prior art, the present inventors constructed a DNA chip by cloning a probe comprising a linker coupled to both ends of about 60 bp target oligonucleotide, into a vector and culturing host cells transformed with the vector, to recover the probe, and integrating the recovered double-helical probe on a substrate. Then, the inventors have found that such a DNA chip showed good signal intensity after hybridization, thereby completing the present invention.